Axonopathy is an early and prominent pathological feature of glaucoma, optic neuritis and traumatic optic nerve injury. Permanent loss of vision in all of these conditions is secondary, in large part, to a failure of retinal ganglion cells (RGC), the output neurons of the optic nerve, to survive and regenerate their axons. There is a dire need to develop novel therapeutic interventions that overcome barriers to repair in the eye, promote RGC survival and RGC axonal regrowth, thereby mitigating, or even reversing, visual loss. In this proposal we investigate a novel subset of neutrophils that accumulate in the vitreous body following intraocular (i.o.) injection of mice with the fungal cell wall extract, zymosan, and are associated with the regrowth of severed RGC axons. In preliminary studies we have demonstrated that adoptive transfer of zymosan-elicited neutrophils directly into the vitreous of mice with optic nerve crush (ONC) injury is sufficient to rescue RGC and stimulate RGC axon regrowth. These neutrophils are characterized by ring-form nuclei and the cell surface phenotype CD14+Ly6Glow. They express high levels of transcripts for arginase-1 and CD206. Our major goals are to elucidate the factors that drive the differentiation of reparative neutrophils and develop protocols to generate them in vitro for therapeutic application in individuals with optic neuropathy. In Aim 1 we will test our hypothesis that IL-4 and granulocyte-colony stimulating factor (G-CSF) act synergistically to drive the differentiation of pro-regenerative neutrophils in vivo in mice subjected to i.o. zymosan injection and ONC injury. A role of IL-4 was suggested by our finding that CD14+Ly6Glow neutrophils express high levels of IL-4 signaling molecules, and IL-4 protein is produced in the vitreous following i.o. administration of zymosan. G- CSF is also upregulated in the vitreous and it was recently shown to induce IL-4 receptor expression on neutrophils. We will determine the kinetics and cellular source of IL-4, IL-4 receptor chains, G-CSF and G-CSF receptor in zymosan injected eyes. Loss and gain of function experiments, using a panel of conditional knock- out mice and bone marrow chimeras, will be performed to assess the roles of IL-4 and G-CSF signaling in the development of CD14+Ly6Glow neutrophils, RGC survival and axonal regeneration following i.o. zymosan injection and ONC. This research could ultimately lead to the development of novel, or the repurposing of established, immunomodulators to promote the differentiation and expansion of neuroregenerative neutrophils in patients with optic neuropathy secondary to glaucoma, optic neuritis or trauma. Our exploratory experiments have shown that murine bone marrow neutrophils acquire characteristics of zymosan-elicited CD14+Ly6Glow neutrophils, including the ability to drive axonal regeneration, following in vitro polarization with IL-4 and G- CSF. The overall goal of Aim 2 is to optimize protocols for the generation of pro-regenerative neutrophils from murine bone marrow precursors ex vivo. The goal of Aim 3 is to assess the neuroregenerative potential of IL-4 modulated human neutrophils that could, potentially, be re-infused into patients with optic neuropathy, as an autologous cell therapy.